Stable vaccine compositions comprising inter alia live attenuated recombinant flavivirus and process for preparation thereof

ABSTRACT

Stable lyophilized immunogenic compositions include inter alia live attenuated recombinant flaviviruses, more preferably live attenuated recombinant dengue viruses, at least one carbohydrate, at least one amino acid and is particularly amenable to rapid freeze-drying treatments wherein, the composition preserves desired characteristics of a virus, including virus viability, immunogenicity and stability. The immunogenic composition is devoid of preservatives, polymers and surfactants. The methods for manufacturing the stable lyophilized immunogenic compositions are also provided.

FIELD OF INVENTION

The present disclosure relates to the field of biotechnology, moreparticularly, it relates to a live attenuated flavivirus vaccinecomposition and the method of preparing the same. The present disclosurefurther relates to an improved methodology in the field of liveattenuated flavivirus vaccine production.

BACKGROUND

The flavivirus genome consists of single stranded, positive sense, RNAmolecule of 11 kilobases, containing single open reading frame. The RNAis translated into a polyprotein that is processed into at least 10 geneproducts: 3 structural proteins—Nucleocapsid or Core (C), Premembrane(prM), & Envelope (E) & 7 non-structural (NS) proteins—NS 1, 2A, 2B, 3,4A, 4B, & 5. (Lindenbach B D, et al., In: Fields Virology. Edited byKnipe D M, Howley P M, Griffin P E, et al. Philadelphia: Wolters Kluwer,Lippencott Williams and Wilkins; 2007. pp. 1101-1152). A number of theseflaviviruses use arthropods (e.g., biting ticks and/or mosquitoes) as ameans for transmission to virus recipients. Such arthropod-borne viruses(i.e., arboviruses) constitute a major worldwide health concern due totheir highly pathogenic nature in humans. (Fernandez-Garcia M D, et al.,Cell Host Microbe, 2009, 5:318-328). More specifically, human arboviruspathogens include yellow fever (YF), Japanese encephalitis (JE), dengue(DEN), West Nile (WN) and tick-borne encephalitis (TBE) viruses thatexist in nature in life cycles which involve mosquito or tick vectorsand avian and/or mammalian competent reservoir hosts. (Gubler D, et al.,In: Fields Virology. Edited by Knipe D M, Howley P M, Griffin P E, etal. 5th ed. Philadelphia: Wolters Kluwer, Lippencott Williams andWilkins; 2007. pp. 1153-1252).

Yet, Dengue virus (DENV) have become the most important human arbovirusworldwide with estimates of as many as 500 million dengue infectionsoccurring annually, resulting in more than 2 million cases of severedisease known as dengue hemorrhagic fever/dengue shock syndrome and21000 deaths. There are four serotypes of dengue virus DENV1 DENV2,DENV3, and DENV4).

Numerous methods are known for producing live attenuated recombinantflavivirus preparations for vaccine and other purposes. Compositions andmethods useful in freezing, lyophilizing, or otherwise storing viablevirus preparations for laboratory or vaccine use in order to preservetheir activity are also known.

The aqueous compositions of flaviviruses do not allow good viralstability in the long term and at a temperature above 5° C. By way ofexample, the bulk aqueous compositions of the YF-DEN (yellowfever-dengue) chimera lose more than 4 log, stabilized in liquid afterstorage for 1 day at 37° C. Now, the thermostability represents aserious problem in subtropical Dengue-endemic countries where transportunder cold-chain conditions is difficult.

Lyophilization is a common mode of stabilization of vaccines. However,lyophilization causes loss in virus potency. Vaccines lose potency overtime and the rate of potency loss is temperature-dependent. Live virusesare susceptible to osmotic, thermal and vacuum shocks. Enveloped virusespossess a lipid bilayer, which is considered as the less stable viruscomponent because of its high fragility. Live viruses are susceptible tovarious stresses during lyophilization steps like freezing, primarydrying, secondary drying that could affect the physico-chemicalstability of viruses. Owing to their structure, loss of potency duringfreeze-drying can be due to protein destabilization (e.g. unfolding,degradation, and aggregation), nucleic acid degradation, lipid layeralteration(e.g. phase transition, mechanical damage) and stressesrelated to changes in the internal (ice formation)and external (pH andosmolarity change) virus environment. The dehydration step oflyophilization results in collapse of the hydrogen bond structure ofproteins which is accompanied with increased mobility of amino acidcomponents of virus epitopes. It has been reported that in some caseslyophilization causes upto 40% loss in virus potency.

Though a lot of information is available on stress mechanisms andstabilization strategies of pharmaceutical peptides, proteins and DNAduring lyophilization, due to the molecular complexity of viruses,different destabilization pathways and lack of analytical techniquespermitting measurement of physico-chemical changes in the antigen'sstructure during and after lyophilization mean that viruses constitute aparticular lyophilization challenge. The destabilization mechanisms aswell as protection mechanisms for live, attenuated viral vaccines duringlyophilization are not well known.

Hansen et al 2015 (Freeze-drying of live virus vaccines: A review,Hansen et al., Vaccine 33 (2015) 5507-5519) discloses a compilation ofseveral freeze dried virus vaccine formulation (s) wherein majority ofthe formulations mention about preferential use of sugar alcohol/proteinadditive (i.e. Sucrose+Trehalose, Sorbitol, Hydrolyzed gelatin,Lactalbumin hydrolysates) for obtaining a lyophilized virus vaccine.

Following flavivirus vaccine formulations have been previouslyreported-1) Sorbitol, Trehalose, Urea, 2) Lactose, Sorbitol, HSA,;3)Lactose, Mannitol, HSA; 4) Poloxamer, Human Albumin , Trehalose, PBS; 5)Trehalose, Recombinant HSA, F127 (polyoxyethylene polyoxypropylene blockcopolymer).

In the case of HSA, the inclusion of these materials may raise potentialsafety concerns if these materials are derived from at-risk human oranimal sources. Such added proteins are of concern for two main reasons.The first concern arises from the potential for animal- andhuman-derived protein to contain one or more adventitious agents. Thesecond concern arises from the potential for animal- or human-derivedprotein to elicit an allergic reaction in susceptible individuals. Also,previously reported lyophilized vaccine formulation uses proteins which,even if produced using processes supporting high yields, have a costimplications for formulations. “For a vaccine to be broadly adopted inlow income regions it is crucial to keep the cost of vaccine and itscomponents such as stabilizers low. It is also crucial from theregulatory and safety point of view that excipients and stabilizers usedshould contain neither substances of animal origin nor contain animalcomponent. Animal-derived compounds represent a potential danger due tothe possible contamination with the scarpie-prion-protein (PrPSC) andthe new variant of the Creutzefeld-Jakob disease(vCJD).

Nonionic surfactants used in pharmaceutical formulations include Triton™X-100, Pluronic® F-68, F-88, and F-127 (poloxamers), Brij 35(polyoxy-ethylene alkyl ether), polyoxyl stearate 40, Cremophor® EL, andalpha-tocopherol TPGS. Each of these surfactants have in common the factthat they all contain polyoxyethylene moieties and thus to a greater orlesser extent, exhibit a similar problem, in that the polyoxyethylenemoiety auto oxidizes to produce reactive peroxides, which causes anincrease in unwanted protein immunogenicity. (Refer Edward T. Maggio etal; Polysorbates, peroxides, protein aggregation, immunogenicity—agrowing concern; Journal of Excipients and Food Chemicals 3(2):46-53;2012).

PVPhas been reported to destabilize live attenuated virus formulations.(Refer: JA

White et al; Development of a stable liquid formulation of liveattenuated influenza vaccine; Vaccine Volume 34, Issue 32, 12 July 2016,Pages 3676-3683; 2016).

Trehalose is costly; it has to be combined with other sugars and proteinadditives (Gelatin) to achieve stability. Also, other stabilizers arebetter than trehalose for enhancing shelf life stability of alyophilized vaccine.

Sorbitol has a low glass-transition temperature (Tg)(−1.6 Deg C.),therefore cannot be used as a main formulation component. The low Tg ofsorbitol limits its use. Sorbitol has to be combined with other sugarsand protein additives (Gelatin) to achieve stability.

Typically, recombinant viruses have been stored as freeze-dried pelletscontaining hydrolysates of casein and/or collagen in phosphate-bufferedphysiological saline (PBS). These pellets are then re-hydrated in apharmaceutically acceptable solution such as 0.4-0.9% NaCl. However,there are significant disadvantages associated with such formulationsand are known in the art. Among these are incompletely definedcomponents, complex preparation procedures, high cost, and inability tomaintain certain desired characteristics of the virus.

Flavivirus vaccine formulations developed previously has been stable at2-8 deg C. for 6 months, 25 deg C. for 7 days and at 37 deg C. for 1-2days. There remains a need for developing formulations that comprise ofminimum number of excipients and impart long term thermostability toflavivirus vaccines, in particular the live attenuatedrecombinant/chimeric Dengue viruses.

Such compositions/formulations and process for preparing of the same aredescribed herein.

SUMMARY

The present disclosure provides an immunogenic composition comprisingablest one live attenuated flavivirus, ablest one carbohydrate, andablest one amino acid wherein, the composition is amenable to rapidfreeze-drying treatments and the reconstituted composition preserves thedesired characteristics of a virus, including virus viability,immunogenicity and stability.

The present disclosure more particularly relates to a lyophilizedimmunogenic compositions comprising:

-   a) Live attenuated recombinant/chimeric dengue virus, wherein the    live attenuated dengue virus strains used are    rDEN1Δ30-1545;rDEN2/4Δ30(ME)-1495, 7163; rDEN3Δ30/31-7164 and    rDEN4Δ30-7132, 7163, 8308 obtained from the United States National    Institutes of Health (NIH).-   b) Sucrose about 3% w/v to about 6% w/v-   c) Glycine about 3% w/v to about 6% w/v

The present disclosure further provides a method for manufacturing suchvaccine composition/formulation.

Objects

Some of the objects of the present disclosure, which at least oneembodiment herein satisfies, are as follows:

An object of the present disclosure is to ameliorate one or moreproblems of the prior art or to at least provide a useful alternative.

Another object of the present disclosure is to provide a stabilizinglyophilized vaccine compositions/formulations comprising of ablest oneflavivirus, ablest one carbohydrate, ablest one amino acid, andoptionally base. Wherein, the composition preserves desiredcharacteristics of a virus, including virus viability, immunogenicityand stability.

Yet another object of the present disclosure is to provide a stabilizinglyophilized vaccine compositions/formulations comprising inter alia alive attenuated recombinant/chimeric dengue virus serotypes (DEN 1, DEN2, DEN 3, DEN 4) suitable for treating or preventing dengue infection,or to prevent, ameliorate, or delay the onset or progression of theclinical manifestations thereof.

Yet another object of the present disclosure is to provide a method formanufacturing such vaccine composition/formulation.

Other objects and advantages of the present disclosure will be moreapparent from the following description, which is not intended to limitthe scope of the present disclosure.

DETAILED DESCRIPTION

Although the present disclosure may be susceptible to differentembodiments, certain embodiments are shown in the figures and followingdetailed discussion, with the understanding that the present disclosurecan be considered an exemplification of the principles of the disclosureand is not intended to limit the scope of disclosure to that which isillustrated and disclosed in this description.

According to a first embodiment of the present disclosure, animmunogenic composition comprising one or more live attenuatedflaviviruses, one or more carbohydrate, and one or more amino acidwherein, the composition is amenable to rapid freeze-drying treatmentsand the reconstituted composition preserves the desired characteristicsof a virus, including virus viability, immunogenicity and stability.

The term “live” is used in its conventional meaning, a live virus is avirus which has not been inactivated, i.e. a virus capable ofreplicating on permissive cells. A live attenuated flavivirus is a viruswhich does not induce the disease caused by the corresponding wild-typevirus in animals or humans and which is capable of inducing a specificimmune response.

According to a second embodiment of the present disclosure, the one ormore live attenuated flaviviruses are a recombinant flaviviruses and/ora chimeric flaviviruses.

According to a third embodiment of the present disclosure, one or morelive attenuated flaviviruses is selected from the group consisting ofdengue (DEN) virus, yellow fever (YF) virus, Japanese encephalitis (JE)virus, Kunjin virus, West Nile (WN) virus, tick-borne encephalitis (TBE)virus, St. Louis encephalitis virus, Murray Valley encephalitis virus,Zika virus, or any related flavivirus thereof.

Yet according to the preferred aspect of the third embodiment, one ormore live attenuated flaviviruses is dengue (DEN) virus, optionally aplurality of live attenuated dengue (DEN) viruses of different serotypesselected from group of DEN-1, DEN-2, DEN-3 and DEN-4.

According to a fourth embodiment of the present disclosure, one or morelive attenuated flaviviruses is selected from the group consisting oflive attenuated chimeric/recombinant yellow fever (YF) viruses and/or ofa live attenuated chimeric/recombinant Japanese encephalitis (JE)viruses, and/or of a live attenuated chimeric/recombinant dengue (DEN)viruses, and/or of a live attenuated chimeric/recombinant West Nile (WN)viruses and/or of a live attenuated chimeric/recombinant tick-borneencephalitis (TBE) viruses and/or of a chimeric dengue virus (yellowfever-dengue) virus, and/or of a chimeric YF-WN (yellow fever-West Nilevirus) virus and/or of a chimeric YF-JE (yellow fever-Japaneseencephalitis) virus or any related flavivirus thereof.

Yet according to a preferred aspect of fourth embodiment, one or morelive attenuated flaviviruses is live attenuated chimeric/recombinantdengue (DEN) viruses.

According to a fifth embodiment of the present disclosure, liveattenuated recombinant/chimeric dengue viruses used in immunogeniccomposition is described below:

A) Brief Description of NIH Recombinant Strains/Its Construction:

All the activities related to generation of attenuated vaccine strainsOf all the four dengue virus serotypes (DEN 1, DEN 2, DEN 3, & DEN 4)explained below have been conducted at NIH, US. Contents of WO2002095075and WO2008022196 are incorporated herein in entirety.

Origin of the Gene

-   1. Each of the attenuated strain of dengue virus serotype 1-4    (rDEN1Δ30, rDEN2/4Δ30(ME), rDEN3Δ30/31 & rDEN4Δ30) has been    developed by deletion of around 30 nucleotides (Δ30) (additional 31    nucleotide (Δ31) in case of DEN-3) from the non-translational 3′ end    of the wild type strains. The Δ31 mutation can also be generated    alone to discern the contribution of either Δ30 or Δ31 in the    combined Δ30/31deletion mutation.-   2. The DEN 2 virus serotype has been developed by replacing the M    and E protein of the attenuated DEN 4 serotype with that of DEN 2 M    and E protein.-   3. Structurally all the four strains are enveloped positive sense    RNA viruses of 35-50 nanometer size.-   4. The rDEN1Δ30-1545 strain used herein encodes a single Lys→Arg    mutation at amino acid residue number 484 (A1545G mutation) in the    viral polyprotein.-   5. The rDEN2/4Δ30(ME)-1495, 7163 strain used herein encodes a    Ser→Phe mutation at amino acid residue number 186 (C1495T mutation)    and a Leu→Phe mutation at amino acid residue number 112 (A7163C    mutation) in the viral polyprotein.-   6. rDEN3Δ30/31 includes the original Δ30 deletion and a    non-contiguous 31 nucleotide deletion that removes both the original    TL-2 and TL-3 structures. The resultant rDEN3Δ30/31-7164 strain used    herein encodes a Val→Ala mutation at amino acid residue number 115    (T7164C mutation) in the viral polyprotein.-   7. The rDEN4Δ30-7132, 7163, 8308 strain used herein encodes a    Thr→Ile mutation at amino acid residue number 102 (C7132T mutation),    a Leu→Phe mutation at amino acid residue number 112 (A7163C    mutation) and a Lys→Arg mutation at amino acid residue number 249    (A8308G mutation) in the viral polyprotein.

Figures depicting the RNA sequence and the virus structure of the DENvaccine strains:

Refer FIGS. 1, 2 and 3

The wild type strains used for the generation of vaccine strains aregiven in Table below:

TABLE 1 Nomenclature of wild type and vaccine strains Serotype Wild typestrain Vaccine strain DEN 1 Western Pacific strain rDEN1Δ30-1545 DEN 2New Guinea strain rDEN2/4Δ30(ME)-1495, 7163 DEN 3 Sleman/78rDEN3Δ30/31-7164 DEN 4 Dominica rDEN4Δ30-7132, 7163, 8308

B) Transformation Procedure:

For the generation of dengue virus vaccine strains essentially thefollowing steps were followed—

-   1. Plasmid containing full length cDNA copy of the wild type DEN    virus was created by generation of short DNA segments using reverse    transcriptase and PCR. Fragments so obtained were appropriately    ligated to generate an intact double stranded DNA comprising of the    full length genomic cDNA strand of the wild type DEN that was cloned    in a plasmid.-   2. Δ30 mutation was inserted by mutating a sub fragment of the 3′UTR    and replacing the 3′UTR of the wild type DEN with the sub fragment    containing the Δ30 region. Specific mutations were introduced by    site specific PCR mutagenesis.-   3. For the generation of DEN 2 vaccine strain structural genes M and    E, of DEN 2 were cloned in plasmid and used to replace the    structural genes in the DEN 4 cloned plasmid containing the Δ30    mutation. For the generation of DEN 3 vaccine strain two deletions    of 30 and 31 nucleotides was introduced in the wild type clone.-   4. Genome length capped RNA transcripts were synthesized from    linearized plasmids using AmpliCap SP6 Message Maker Kit (EpiCentre    Technologies, Madison) and the RNA purified using the RNeasy Mini    kit (Qiagen, Valencia, Calif.).

Vero cells (C6/36 for dengue 3) were transfected with purified RNAtranscripts using DOTAP liposomal transfection reagents (Roche,Indianapolis, Ind.) to recover desired virus. Rescued viruses weresubjected to amplification, terminal dilution cloning and finalamplification for the generation of seed virus in Vero cells. Details onnumber of cycles of amplification and terminal dilution undertaken foreach strain are tabulated below in table 2.

TABLE 2 Cycle of amplification and terminal dilution for seed viruspreparation Virus strain DEN 1 DEN 2 DEN 3 DEN 4 Rescued in Vero VeroC6/36* Vero Amplification Nil Nil 6X 3X Terminal dilution 2X 2X 3X 3Xcloning Amplification 2X 2X 2X 2X *All further work of amplification andterminal dilution cloning was carried out in Vero cells.

According to a first aspect of the fifth embodiment, the chimericviruses have the particularity of exhibiting the characteristics of thelive attenuated viruses as defined above. It is therefore possible touse, in the context of the disclosure, any chimeric virus expressing theenvelope protein or one or more epitopes of one or more envelopeprotein(s) of one or more flaviviruses and inducing a specific immuneresponse comprising antibodies which neutralize the strain, or at leastone of the strains, from which the envelope protein or said epitope isderived.

According to a second aspect of the fifth embodiment, the liveattenuated recombinant dengue virus nucleic acid further comprises amutation generating a mutant having a phenotype selected from the groupconsisting of temperature sensitivity in Vero cells or the human livercell line HuH-7, host-cell restriction in mosquito cells or the humanliver cell line HuH-7, host-cell adaptation for improved replication inVero cells, or attenuation in mice or monkeys, wherein the compositioncomprising a member selected from the group consisting of:

(1) rDENIΔ30, rDEN2Δ30, rDEN3Δ30, rDEN4Δ30,

(2) rDENIΔ30, rDEN2Δ30, rDEN3Δ30, rDEN4/1Δ30,

(3) rDENIΔ30, rDEN2Δ30, rDEN3Δ30, rDEN4/2Δ30,

(4) rDENIΔ30, rDEN2Δ30, rDEN3Δ30, rDEN4/3Δ30,

(5) rDENlΔ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4Δ30,

(6) rDENIΔ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(7) rDENIΔ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(8) rDENIΔ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(9) rDENIΔ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4Δ30,

(10) rDENIΔ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(11) rDENIΔ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(12) rDENIΔ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(13) rDENIΔ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4Δ30,

(14) rDENIΔ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(15) rDEMΔ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(16) rDENIΔ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(17) rDENIΔ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4Δ30,

(18) rDENIΔ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/IΔ30,

(19) rDENIΔ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/2Δ30,

(20) rDENIΔ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/3Δ30,

(21) rDENIΔ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4Δ30,

(22) rDENIΔ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/IΔ30,

(23) rDENIΔ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/2Δ30,

(24) rDENIΔ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/3Δ30,

(25) rDENIΔ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4Δ30,

(26) rDENIΔ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/IΔ30,

(27) rDEMΔ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/2Δ30,

(28) rDENIΔ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/3Δ30,

(29) rDENIΔ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4Δ30,

(30) rDENIΔ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/IΔ30,

(31) rDENIΔ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/2Δ30,

(32) rDENIΔ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/3Δ30,

(33) rDENIΔ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4Δ30,

(34) rDENIΔ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/IΔ30,

(35) rDENIΔ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/2Δ30,

(36) rDENIΔ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/3Δ30,

(37) rDENIΔ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4Δ30,

(38) rDENIΔ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(39) rDENIΔ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(40) rDENIΔ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(41) rDENIΔ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4Δ30,

(42) rDENIΔ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(43) rDENIΔ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(44) rDENIΔ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(45) rDENIΔ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4Δ30,

(46) rDENIΔ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(47) rDENIΔ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(48) rDENIΔ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(49) rDENIΔ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4Δ30,

(50) rDENIΔ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/IΔ30,

(51) rDENIΔ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/2Δ30,

(52) rDENIΔ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/3Δ30,

(53) rDENIΔ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4Δ30,

(54) rDENIΔ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(55) rDENIΔ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(56) rDENIΔ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(57) rDENIΔ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4Δ30,

(58) rDENIΔ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(59) rDENIΔ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(60) rDENIΔ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(61) rDENIΔ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4Δ30,

(62) rDENIΔ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(63) rDENIΔ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(64) rDENIΔ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(65) rDENI/2Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4Δ30,

(66) rDENI/2Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/IΔ30,

(67) rDENI/2Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/2Δ30,

(68) rDENI/2Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/3Δ30,

(69) rDENI/2Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4Δ30,

(70) rDENI/2Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(71) rDENI/2Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(72) rDENI/2Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(73) rDENI/2Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4Δ30,

(74) rDENI/2Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(75) rDENI/2Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(76) rDENI/2Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(77) rDENI/2Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4Δ30,

(78) rDENI/2Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(79) rDENI/2Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/2Δ30, i

(80) rDENI/2Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(81) rDENI/2Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4Δ30,

(82) rDENI/2Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/IΔ30,

(83) rDENI/2Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/2Δ30,

(84) rDENI/2Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/3Δ30,

(85) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4Δ30,

(86) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/IΔ30,

(87) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/2Δ30,

(88) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/3Δ30,

(89) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4Δ30,

(90) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/IΔ30,

(91) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/2Δ30,

(92) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/2Δ30₅ rDEN4/3Δ30,

(93) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4Δ30,

(94) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/IΔ30,

(95) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/2Δ30,

(96) rDENI/2Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/3Δ30,

(97) rDENI/2Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4Δ30,

(98) rDENI/2Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/IΔ30,

(99) rDENI/2Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/2Δ30, _(I)

(100) rDENI/2Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/3Δ30,

(101) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4Δ30,

(102) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(103) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(104) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(105) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4Δ30,

(106) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(107) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(108) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(109) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4Δ30,

(110) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(111) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(112) rDENI/2Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(113) rDENI/2Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4Δ30,

(114) rDENI/2Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/IΔ30,

(115) rDENI/2Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/2Δ30,

(116) rDENI/2Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/3Δ30,

(117) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4Δ30,

(118) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(119) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(120) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(121) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/2Δ30₅ rDEN4Δ30,

(122) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(123) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(124) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(125) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4Δ30,

(126) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(127) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(128) rDENI/2Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(129) rDENI/3Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4Δ30,

(130) rDENI/3Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/IΔ30,

(131) rDENI/3Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/2Δ30,

(132) rDENI/3Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/3Δ30,

(133) rDENI/3Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4Δ30,

(134) rDENI/3Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(135) rDENI/3Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(136) rDENI/3Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(137) rDENI/3Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4Δ30,

(138) rDENI/3Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(139) rDENI/3Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(140) rDENI/3Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(141) rDENI/3Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4Δ30,

(142) rDENI/3Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(143) rDENI/3Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(144) rDENI/3Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(145) rDENI/3Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4Δ30,

(146) rDENI/3Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/IΔ30,

(147) rDENI/3Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/2Δ30,

(148) rDENI/3Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/3Δ30,

(149) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4Δ30,

(150) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/IΔ30,

(151) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/2Δ30,

(152) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/3Δ30,

(153) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4Δ30,

(154) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/IΔ30,

(155) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/2Δ30,

(156) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/3Δ30,

(157) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4Δ30,

(158) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/IΔ30,

(159) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/2Δ30,

(160) rDENI/3Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/3Δ30,

(161) rDENI/3Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4Δ30,

(162) rDENI/3Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/IΔ30,

(163) rDENI/3Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/2Δ30,

(164) rDENI/3Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/3Δ30,

(165) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4Δ30,

(166) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(167) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(168) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(169) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4Δ30,

(170) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(171) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(172) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(173) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4Δ30,

(174) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(175) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(176) rDENI/3Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(177) rDENI/3Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4Δ30,

(178) rDENI/3Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/IΔ30,

(179) rDENI/3Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/2Δ30,

(180) rDENI/3Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/3Δ30,

(181) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4Δ30,

(182) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(183) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(184) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(185) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4Δ30,

(186) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(187) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(188) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(189) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4Δ30,

(190) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(191) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(192) rDENI/3Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(193) rDENI/4Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4Δ30,

(194) rDENI/4Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/IΔ30,

(195) rDENI/4Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/2Δ30,

(196) rDENI/4Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/3Δ30,

(197) rDENI/4Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4Δ30,

(198) rDENI/4Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(199) rDENI/4Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(200) rDENI/4Δ30, rDEN2Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(201) rDENI/4Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4Δ30,

(202) rDENI/4Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(203) rDENI/4Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(204) rDENI/4Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(205) rDENI/4Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4Δ30,

(206) rDENI/4Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/1130,

(207) rDENI/4Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(208) rDENI/4Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(209) rDENI/4Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4Δ30,

(210) rDENI/4Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/IΔ30,

(211) rDENI/4Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/2Δ30,

(212) rDENI/4Δ30, rDEN2/IΔ30, rDEN3Δ30, rDEN4/3Δ30,

(213) rDENI/4Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4Δ30,

(214) rDENI/4Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/IΔ30,

(215) rDENI/4Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/2Δ30,

(216) rDENI/4Δ30, rDEN2/IΔ30, rDEN3/IΔ30, rDEN4/3Δ30,

(217) rDENI/4Δ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4Δ30,

(218) rDENI/4Δ30₅rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/IΔ30,

(219) rDENI/4Δ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/2Δ30,

(220) rDENI/4Δ30, rDEN2/IΔ30, rDEN3/2Δ30, rDEN4/3Δ30,

(221) rDENI/4Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4Δ30,

(222) rDENI/4Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/IΔ30,

(223) rDENI/4Δ30, rDEN2/IA305 rDEN3/4Δ30, rDEN4/2Δ30,

(224) rDENI/4Δ30, rDEN2/IΔ30, rDEN3/4Δ30, rDEN4/3Δ30,

(225) rDENI/4Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4Δ30,

(226) rDENI/4Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/IΔ30,

(227) rDENI/4Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/2Δ30,

(228) rDENI/4Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/3Δ30,

(229) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4Δ30,

(230) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(231) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(232) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(233) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4Δ30,

(234) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(235) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(236) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(237) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4Δ30,

(238) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(239) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(240) rDENI/4Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(241) rDENI/4Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4Δ30,

(242) rDENI/4Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/IΔ30,

(243) rDENI/4Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/2Δ30,

(244) rDENI/4Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/3Δ30,

(245) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4Δ30,

(246) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/IΔ30,

(247) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/2Δ30,

(248) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/IΔ30, rDEN4/3Δ30,

(249) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4Δ30,

(250) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/IΔ30,

(251) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(252) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(253) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4Δ30,

(254) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/IΔ30,

(255) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

and

(256) rDENI/4Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/3Δ30.

According to sixth embodiment of the present disclosure, one or morecarbohydrates include, but are not limited to, natural carbohydrates,synthetic carbohydrates, polyols, glass transition facilitating agentsmonosaccharides, disaccharides, trisaccharides, oligosaccharides andtheir corresponding sugar alcohols, polyhydroxyl compounds such ascarbohydrate derivatives and chemically modified carbohydrates,hydroxyethyl starch and sugar copolymers. Both natural and syntheticcarbohydrates are suitable for use. Synthetic carbohydrates include, butare not limited to, those which have the glycosidic bond replaced by athiol or carbon bond. Both D and L forms of the carbohydrates may beused. The carbohydrate may be non-reducing or reducing. Where a reducingcarbohydrate is used, the addition of inhibitors of the Maillardreaction is preferred. Reducing carbohydrates suitable for use in thecomposition are those known in the art and include, but are not limitedto, glucose, sucrose, maltose, lactose, fructose, galactose, mannose,maltulose and lactulose. Non-reducing carbohydrates include, but are notlimited to, non-reducing glycosides of polyhydroxyl compounds selectedfrom sugar alcohols and other straight chain polyalcohols. Other usefulcarbohydrates include raffinose, stachyose, melezitose, dextran,cellibiose, mannobiose and sugar alcohols. The sugar alcohol glycosidesare preferably monoglycosides, in particular the compounds obtained byreduction of disaccharides such as lactose, maltose, lactulose andmaltulose. Glass forming agent is selected from the group consisting ofsucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionicacid, glucose, maltulose, iso-maltulose, maltose, lactose sorbitol,dextrose, fucose or a combination thereof.

Yet according to the preferred aspect of the sixth embodiment, animmunogenic composition comprises of sucrose as suitable carbohydratestabilizer ranging in between 1% and 20% weight/volume, preferably inbetween 1-10%, more preferably in between 3-6%, most preferably lessthan or equal to 5% (w/v).

According to seventh embodiment of the present disclosure, the one ormore amino acid include, but are not limited to, leucine, iso-leucine,histidine, glycine, glutamine, arginine, lysine, alanine or acombination of amino acids, peptide, hydrolysed protein or protein suchas serum albumin.

Yet according to the preferred aspect of the seventh embodiment, animmunogenic composition comprises of glycine as suitable amino acidstabilizer ranging in between 1% and 20% weight/volume, preferably inbetween 1-10%, more preferably in between 3-6%,most preferably less thanor equal to 5% (w/v).

According to a eighth embodiment of the present disclosure, animmunogenic composition may additionally comprise of a buffering agentselected from the group consisting of carbonate, phosphate, citrate,lactate, gluconate and tartrate buffering agents, as well as morecomplex organic buffering agents including a phosphate buffering agentthat contains sodium phosphate and/or potassium phosphate in a ratioselected to achieve the desired pH. In another example, the bufferingagent contains Tris (hydroxymethyl) aminomethane, or “Tris”, formulatedto achieve the desired pH. Yet in another example, the buffering agentcould be the minimum essential medium with Hanks salts.

According to a ninth embodiment of the present disclosure, animmunogenic composition may additionally comprise of preservativeselected from the group consisting of 2-phenoxyethanol, Benzethoniumchloride (Phemerol), Phenol, m-cresol, Thiomersal, Formaldehyde, methyland propyl parabens, benzalkonium chloride, benzyl alcohol,chlorobutanol, p-chlor-m-cresol, or benzyl alcohol or a combinationthereof.

According to a tenth embodiment of the present disclosure, animmunogenic composition may additionally comprise of pharmaceuticallyacceptable excipients selected from the group consisting of surfactants,polymers and salts. Examples of Surfactants may include non-ionicsurfactants such as polysorbate 20, polysorbate 80, etc. Examples of thepolymers may include dextran, carboxymethylcellulose, hyaluronic acid,cyclodextrin, etc. Examples of the salts may include NaCl, MgC12, KCl,CaC12, etc.

According to an eleventh embodiment of the present disclosure, animmunogenic composition may additionally comprise of an adjuvantselected from the group consisting of an aluminum salt, aluminumhydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassiumaluminum sulfate.

According to twelfth embodiment of the present disclosure, animmunogenic composition may additionally comprise of animmunostimulatory component selected from the group consisting of: anoil and water emulsion, MF-59,a liposome, a lipopolysaccharide, asaponin, lipid A, lipid A derivatives, Monophosphoryl lipid A,3-deacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, anoligonucleotide comprising at least one unmethylated CpG and/or aliposome, Freund's adjuvant, Freund's complete adjuvant, Freund'sincomplete adjuvant, polymers, co-polymers such aspolyoxyethylene-polyoxypropylene copolymers, including blockco-polymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, TLR-4agonists, flagellin, flagellins derived from gram negative bacteria,TLR-5 agonists, fragments of flagellins capable of binding to TLR-5receptors, QS-21, ISCOMS, saponin combination with sterols and lipids.

According to thirteenth embodiment of the present disclosure, the saidimmunogenic composition is lyophilized (freeze-dried).

According to a fourteenth embodiment of the present disclosure, thelyophilized immunogenic composition is stable at 2-8 deg C. from 12 to36 months; at 25 deg C. from 2 to 6 months; at 37 deg C. from 1 week to4 weeks, at 42 deg C. for 2-7 days, at 55 deg C. for 2-7 days.

According to fifteenth embodiment of the present disclosure, a methodfor reconstituting a lyophilized immunogenic composition comprising thestep of reconstituting the lyophilized immunogenic composition with anaqueous solution optionally saline or water for injection(WFI).

According to sixteenth embodiment of the present disclosure, the finalpH of the immunogenic composition after reconstitution is in the rangeof pH 6.0 to pH 8.0; more preferably in the range of pH 7.0 to pH 8.0;more preferably in the range of pH 7.2 to pH 7.9; and most preferably inthe range of pH 7.5 to pH 7.9.

According to a seventeenth embodiment of the present disclosure, theprocess for preparing live attenuated chimeric/recombinant tetravalentdengue(DEN) vaccine composition comprises any subset or all of thefollowing steps:

-   a) Vero cells were revived and adapted to grow in Minimum essential    medium (MEM) with Hank's salt solution and 10% Fetal bovine serum-   b) Vero cells were initially amplified in tissue culture flasks (TCF    with 175 cm² surface area available for cell growth) producing    Master banks and Working banks of Vero cells-   c) Cryopreserved cells from the working cell bank were revived,    amplified and further passaged in roller bottles (850 cm² surface    area available for cell growth) and incubated at 37±1° C. to obtain    monolayers-   d) Vero cell monolayers in roller bottles were infected with working    seed of dengue virus serotypes 1, 2, 3 and 4-   e) All roller bottles were incubated at 34±1° C. for 20 min.; and    volume top up to 120 ml per RB using Minimum essential medium (MEM)    with Hanks salt solution and 2% Fetal bovine serum. Further all    roller bottles were incubated at 34±1° C. for 2 days and 0.7RPM    rolling speed.-   f) On day 2, monolayers in roller bottles were washed with fresh    virus medium devoid of fetal bovine serum and RBs were incubated at    34±1° C. for 3 dayseach at 0.7 RPM rolling speed-   g) On 5^(th) day post infection the cell supernatant from all the    infected roller bottles was harvested and bottles re-fed with fresh    virus medium devoid of fetal bovine serum;-   h) Multiple harvests were taken and processed separately to obtain    clarified monovalent virus pools (CMVPs)-   i) Filtering the viral harvest by direct flow filtration (DFF)    through at least one clarification filter-   j) Treating the viral harvest with a non-specific endonuclease to    degrade cellular DNA-   k) The treated viral harvest was subjected to tangential flow    filtration-   l) Stabilizing the viral harvest with a stabilizing agent comprising    of ablest one amino-acid and ablest one carbohydrateto form a    stabilized viral harvest-   m) Sterilizing the stabilized viral harvest by DFF through at least    one sterilization grade filter-   n) Clarified monovalent virus pools (CMVPs) of each of the dengue    virus serotype were stored in polycarbonate bottles at −60° C. or    below-   o) Clarified monovalent virus pools (CMVPs)of all four virus    serotypes were mixed together to obtain final bulk which is filled    in vials and lyophilized to obtain the drug product i.e recombinant    dengue tetravalent vaccine (live attenuated)

According to a first aspect of seventeenth embodiment, the Vero cellline used were ATCC CCL-81 (cGMPVero, Kidney cells derived from Africangreen monkey (Cercopithecus aeothiops; available from the ATCC,Manassas, Va., USA)

According to a second aspect of seventeenth embodiment, multipleharvests were carried out at an appropriate time interval for about 4-5times—more preferably 4 times on 5^(th) Day, 7^(th) Day, 9^(th) Day &11^(th) Day before discarding the input material and processedseparately to obtain clarified monovalent virus pools (CMVPs). In caseof multiple harvests the same quantity of input material contributeshigher yield as compared to conventional single harvest method. Thisalso saves time and total production cost for upstream processing i.e.amplification of cells for infection.

According to a third aspect of seventeenth embodiment, wherein the virusmedium comprises of Minimum Essential Medium (MEM) with Hanks saltsolution additionally containing Dextrose, L-Glutamine and SodiumBicarbonate.

According to a fourth aspect of seventeenth embodiment, the mediumcontaining the virus is clarified, typically through filters ofdecreasing pore sizes (e.g., 6μ, 0.8μ, 0.45μ, 0.2μ). Suitablecommercially available filters and filtration devices are well known inthe art and can be selected by those of skill. Exemplary filtrationdevices include, e.g., Millipak (Millipore), Kleenpak (Pall) andSartobran™ P filtration devices.

According to a fifth aspect of seventeenth embodiment, the filteredharvest was treated with a non-specific endonuclease most preferablyBenzonase with concentration varying in between 1-10 units/ml, attemperature ranging in between 4-37° C.,and for intervals ranging inbetween 2 hours to 12 hours.

According to a sixth aspect of seventeenth embodiment, the Benzonasetreated harvest was further subjected to tangential flow filtration(TFF) typically through filters with a molecular weight cut off (MWCO)of 500 KD, more preferably 300 KD and most preferably 100 KD.

According to seventh aspect of the seventeenth embodiment, the viralharvest was subjected to tangential flow filtration (TFF) resulting inat least 10× concentration of viral harvest and further results in theremoval of residual impurities.

Yet preferable the residual impurities comprises of residual DNA,residual bovine serum albumin (BSA) and residual host cell protein.

According to eighth aspect of the seventeenth embodiment, the processdescribed above result in a purified and concentrated flaviviruspreparation more preferably dengue virus preparation wherein, thepreparations comprises of concentrated live attenuated dengue virusparticles, traces of residual cellular DNA (<10 ng/dose), residual BSA(<50 ng/dose) and residual cellular proteins. Furthermore, according tothe process described above, the overall recovery of purified viruses isat least 50%.

According to ninth aspect of the seventeenth embodiment, stabilizerscomprising solution of one or more amino-acid and one or morecarbohydrate were mixed with concentrated virus stock (TFF concentrate)in 60:40 or 50:50 or 40:60 proportion of virus stock to stabilizer toobtain the final formulation.

Yet preferable the stabilizers comprising solution of sucrose at aconcentration of 7.5 to 15% (w/v) and glycine at a concentration of 7.5to 15% (w/v) were mixed with concentrated virus stock (TFF concentrate)in 60:40 or 50:50 or 40:60 proportion of virus stock to stabilizer toobtain the final formulation comprising sucrose at a concentration of 3to 6% (w/v) and glycine at a concentration of 3 to 6% (w/v).

Yet preferable the stabilizers comprising solution of sucrose at aconcentration of 12.5% (w/v) and glycine at a concentration of 12.5%(w/v) were mixed with concentrated virus stock (TFF concentrate) in60:40 proportion of virus stock to stabilizer to obtain the finalformulation comprising sucrose at a concentration of 5% (w/v) andglycine at a concentration of 5% (w/v).

Yet preferable the stabilizers comprising solution of sucrose at aconcentration of 11.25% (w/v) and glycine at a concentration of 12.5%(w/v) were mixed with concentrated virus stock (TFF concentrate) in60:40 proportion of virus stock to stabilizer to obtain the finalformulation comprising sucrose at a concentration of 4.5% (w/v) andglycine at a concentration of 5% (w/v).

Yet preferable the stabilizers comprising solution of sucrose at aconcentration of 15% (w/v) and glycine at a concentration of 15% (w/v)were mixed with concentrated virus stock (TFF concentrate) in 60:40proportion of virus stock to stabilizer to obtain the final formulationcomprising sucrose at a concentration of 6% (w/v) and glycine at aconcentration of 6% (w/v).

According to tenth aspect of seventeenth embodiment, the multiplicity ofinfection (MOI) of flavivirus more preferably dengue virus to obtainmaster seed and working seed is in the range 0.01 to 0.1 for each dengueserotype.

Yet preferably the multiplicity of infection (MOI) of dengue virus toobtain master seed and working seed is 0.01.

According to a eleventh aspect of seventeenth embodiment, theimmunogenic composition comprises flavivirus more preferably denguevirus at a dose of not less than 2.5 log₁₀ PFU per 0.5 ml of each ofdengue virus serotype 1, 2, 3 and 4

According to a twelfth aspect of seventeenth embodiment, the immunogeniccomposition comprises dengue virus at a dose of log 10³ to log 10⁵ PFUper 0.5 ml, more preferably log 10³ to log 10⁴ PFU/per 0.5 ml, mostpreferably log 10³ PFU/per 0.5 ml of each of dengue virus serotype 1, 2,3 and 4.

According to an eighteenth embodiment of the present disclosure, themethod of lyophilization (freeze-drying) of an immunogenic compositioncomprises the steps of freezing, primary drying and secondary drying.

Yet preferably the method of lyophilization (freeze-drying) of liveattenuated chimeric/recombinant tetravalent dengue (DEN) vaccinecomposition comprises any subset or all of the following steps:

-   a) Product loading at temperatures between 20 to 5° C.-   b) Stepwise freezing with holding time at each temperature, wherein    freezing step comprises of freezing at about −30° C. to −45° C. at a    freezing rate of 0.5 to 1° C./min from about 60 minutes to 930    minutes-   c) Annealing at −20° C. for 5 hrs followed by freezing at −45° C.-   d) Primary drying comprises stepwise ramping of temperature at about    0.5° C./minute to 1.0° C./minute to achieve shelf temperature of    about −25° C. to −32° C. holding for about 600 minutes to 1980    minutes under pressure of about 55 pbar;-   e) Secondary drying involve heating product at the rate of 0.5 to    1.0° C./min, to achieve shelf temperature of about 25° C. to 30° C.    holding for about 360 minutes to 600 minutes under pressure of 55    pbar.

The total duration of the lyophilization cycle comprises from about 48hours to 56 hours. Variations in temperature and cycle duration as pervial specification and lyophilizer design are contemplated. The productis lyophilized based on a pre-determined cycle to achieve a targetmoisture content of about 2.0% w/w to 3.5% w/w.

As used herein the terms “Freeze-drying” or “lyophilize” or“lyophilization” involves lyophilization and refers to the process bywhich a suspension is frozen, after which the water is removed bysublimation at low pressure. As used herein, the term “sublimation”refers to a change in the physical properties of a composition, whereinthe composition changes directly from a solid state to a gaseous statewithout becoming a liquid.

According to a nineteenth embodiment of the present disclosure, theimmunogenic composition is formulated for use in a method for reducingthe onset of or preventing a health condition involving administrationof an effective amount of the immunogenic composition to a human subjectvia intramuscular, or intravenous, subcutaneous, or transcutaneous orintradermal.

According to a twentieth embodiment of the present disclosure, thehealth condition is selected from the group consisting of Dengue virusinfection, Zika virus infection, West Nile infection, Japaneseencephalitis infection, Kunjin virus infection, tick-borne encephalitisinfection, St. Louis encephalitis virus infection, Murray Valleyencephalitis virus infection, yellow fever virus infection.

According to a twenty first embodiment of the present disclosure, theimmunogenic composition may be administered subcutaneously,intradermally, or intramuscularly in a dose effective for the productionof neutralizing antibody and protection. The vaccines are administeredin a manner compatible with the dosage formulation, and in such amountas will be prophylactically and/or therapeutically effective. Theimmunogenic composition of the present disclosure can be administered asprimary prophylactic agents in adults or children at the risk ofinfection, or can be used as secondary agents for treating infectedpatients. For example, the live attenuated dengue (DEN) tetravalentvaccine composition as disclosed herein can be used in adults orchildren at risk of dengue virus infection, or can be used as secondaryagents for treating DEN virus infected patients.

According to a twenty second embodiment of the present disclosure, theimmunogenic composition can be formulated as single dose vials,multidose vials or as pre-filled syringes wherein the said immunogeniccomposition may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of vaccination may bewith 1-2 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the immuneresponse, for example, at 1-4 months for a second dose, and if needed, asubsequent dose(s) after several months or years. The dosage regimenwill also, at least in part, be determined on the need of a booster doserequired to confer protective immunity.

Other embodiments disclosed herein also encompasses vaccine kitcomprising a first container containing a lyophilized(freeze-dried)immunogenic composition and a second container containingan aqueous solution optionally saline or WFI (water for injection) forthe reconstitution of the lyophilized (freeze-dried) immunogeniccomposition.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps and can mean “includes,”“including,” and the like; “consisting essentially of” or “consistsessentially” likewise has the meaning ascribed in U.S. Patent law andthe term is open-ended, allowing for the presence of more than thatwhich is recited so long as basic or novel characteristics of that whichis recited is not changed by the presence of more than that which isrecited, but excludes prior art embodiments.

Throughout this specification the word, “immunogenic composition” coversany composition that elicits an immune response against the antigen orimmunogen of interest expressed from vectors; for instance, afteradministration into a subject, elicits an immune response against thetargeted immunogen or antigen of interest. The terms “vaccinecomposition” and “vaccine” covers any composition that induces aprotective immune response against the antigen of interest, or whichefficaciously protects against the antigen; for instance, afteradministration or injection into the subject, elicits a protectiveimmune response against the targeted antigen or immunogen or providesefficacious protection against the antigen or immunogen expressed fromvectors.

The use of the expression “at least” or “at least one” suggests the useof one or more elements or ingredients or quantities, as the use may bein the embodiment of the invention to achieve one or more of the desiredobjects or results. While certain embodiments of the inventions havebeen described, these embodiments have been presented by way of exampleonly, and are not intended to limit the scope of the inventions.Variations or modifications to the formulation of this invention, withinthe scope of the invention, may occur to those skilled in the art uponreviewing the disclosure herein. Such variations or modifications arewell within the spirit of this invention.

The numerical values given for various physical parameters, dimensionsand quantities are only approximate values and it is envisaged that thevalues higher than the numerical value assigned to the physicalparameters, dimensions and quantities fall within the scope of theinvention unless there is a statement in the specification to thecontrary.

Similarly, the components used in purification, e.g., filters, columns,are not intended to be in any way limiting or exclusionary, and can besubstituted for other components to achieve the same purpose at thediscretion of the practitioner.

While considerable emphasis has been placed herein on the specificfeatures of the preferred embodiment, it will be appreciated that manyadditional features can be added and that many changes can be made inthe preferred embodiment without departing from the principles of thedisclosure. These and other changes in the preferred embodiment of thedisclosure will be apparent to those skilled in the art from thedisclosure herein, whereby it is to be distinctly understood that theforegoing descriptive matter is to be interpreted merely as illustrationof the disclosure and not as a limitation.

Advantages

The present disclosure described herein above has several technicaladvances and advantages including, but not limited to, the realizationof a stable lyophilized immunogenic composition comprising liveattenuated recombinant dengue viruses, ablest one carbohydrate, ablestone amino acid and the method of manufacturing the same. When comparedto other lyophilized immunogenic composition, the present disclosureprovides the following advantages:

1. Minimum components involved in the vaccine composition.

2. The reconstituted vaccine preserves desired characteristics of avirus including virus viability, immunogenicity and stability.

3. Improved stability at 2-8° C., 25° C., 37° C., 42° C. and 55° C. foran extended period.

4. Devoid of preservatives, polymers and surfactants.

5. Improved method of manufacturing such stable composition/formulationthat result in improved yield. Examples:

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the compositions and techniques disclosed in the examples whichfollow represent techniques discovered by the inventor to function wellin the practice disclosed herein, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

EXAMPLE 1

Multiple Harvesting vs. Single Harvest

Certain experiments were performed to initially identify the method ofmanufacturing the immunogenic composition suitable for preclinical andclinical testing and use of flavivirus immunogenic compositions orvaccines were identified. In some exemplary methods, live, attenuatedrecombinant/chimeric dengue viruses were used as an exemplaryflaviviruses in various compositions for pre-clinical and clinicaltesting. The candidate dengue vaccine strains were supplied by NationalInstitute of Health (NIH), USA.

The process for manufacturing live attenuated chimeric/recombinanttetravalent dengue (DEN) vaccine composition comprises any subset or allof the following steps:

-   1. Vero cells were revived and adapted to grow in Minimum essential    medium (MEM) with Hanks salt solution and 10% Fetal bovine serum    (FBS);-   2. Vero cells were initially amplified in tissue culture flasks (TCF    with 175 cm² surface area available for cell growth) producing    Master banks and Working banks of Vero cells.-   3. Cryopreserved cells from the working cell bank were revived,    amplified and further passaged in roller bottles (RBs) (850 cm²    surface area available for cell growth) and incubated at 37±1° C. &    0.7 RPM rolling speed to obtain monolayers.-   4. Vero cell monolayers in roller bottles were infected with working    seed of dengue virus serotypes 1, 2, 3 and 4 at 0.01 MOI-   5. All roller bottles were incubated at 34±1° C. for 20 min.; and    volume top up to 120m1 per RB using Minimum essential medium (MEM)    with Hanks salt solution and 2% Fetal bovine serum. Further all    roller bottles were incubated at 34±1° C. for 2 days and 0.7RPM    rolling speed.-   6. On day 2 monolayers in roller bottles were washed with fresh    virus medium devoid of fetal bovine serum to remove traces of FBS    and were incubated at 34±1° C. & 0.7 RPM rolling speed for 3 days.-   7. On 5th day post infection the cell supernatant from all the    infected roller bottles was harvested and bottles re-fed with fresh    virus medium comprising MEM with Hanks salt solution additionally    containing Dextrose, L-Glutamine and Sodium Bicarbonate and is    devoid of fetal bovine serum;

TABLE 3 Composition of Minimum Essential Medium with Hank's saltComponent MG/L Calcium Chloride (Anhydrous) 140 Magnesium Sulphate(Anhydrous) 98 Potassium Chloride 400 Potassium Phosphate Monobasic(Anhydrous) 60 Sodium Chloride 8000 Sodium Phosphate Dibasic (Anhydrous)48 L-Arginine HCl 126 L-Cystine HCl 31 L-Glutamine 292 L-Histidine HClH20 42 L-Isoleucine 52 L-Leucine 52 L-Lysine HCl 73 L-Methionine 15L-Phenylalanine 32 L-Threonine 48 L-Tryptophan 10 L-Tyrosine 2NA 2H2O 52L-Valine 46 Choline Chloride 1 Folic Acid 1 I-Inositol 2 Niacinamide 1D-Panthothenic Acid (Hemicalcium) 1 Pyridoxal HCl 1 Riboflavin 0.1Thiamine HCl 1 Glucose 1000 Phenol Red 10

Various approaches for harvesting of dengue virus were carried out assingle harvest and multiple harvests at various time points (from day4to day12 after infection) as daily, on odd days, on even days etc. Thecomparison of virus titers with respect to yields by single harvestversus multiple harvests was conducted.

TABLE 4 Harvesting Time Points Set No. No. of RBs Harvest/SamplingDetails 1 3 Daily harvest on 4, 5, 6, 7, 8, 9 days 2 3 Even day harveston 4, 6, 8 days 3 3 Odd day harvest on 5, 7, 9, 11 days. 4 3 Dailysampling on 4, 5, 6, 7, 8, 9, 10, 11 days 5 3 Single harvest on day 6 63 Single harvest on day 7

From day 4, the infected RBswere harvested set wise (set 1 to 4) onrespective days. After harvesting, the RBs were re-fed with fresh virusmedium-VM, and incubated at 34° C. till next harvest. Also a singleharvest of supernatant was collected on day 6 (set 5) & day 7 (set 6)respectively. These samples were tested for virus titers (CCID₅₀) bySpearman Karber method.

TABLE 5 DEN 1 Virus Titers (CCID50) DEN 1 Virus Titers (CCID₅₀/ml)Harvest Odd Even Single Growth Days Day Day Daily Harvest Curve Day 45.5 5.5 6 Day 5 6.125 6.375 6.3 Day 6 6.625 6.375 6.975 7 Day 7 7.375 77.1 7.25 Day 8 7.125 6.625 7.625 Day 9 7.25 6.5 7 Day 10 6.5 6.125 6.875Day 11 6.25 6.5 7 Day 12 6.25

The growth curve obtained with Dengue Virus Serotype 1 (DEN 1) showedthat multiple harvesting on day 5, 7, 9 & 11 gave good virus titers andhence would be the choice for further batches (Refer FIG. 4).

One more trial on multiple harvests versus single harvest was conductedusing 18RBs for Den 2, 3 & 4.

TABLE 6 DEN 2, 3, 4 Virus Titers (CCID₅₀) Harvest Virus Titers(CCID₅₀/ml) Days DEN 2 DEN 3 DEN 4 Multiple Day 5 6.621 5.825 7.432Harvest Day 7 6.73 6.021 7.588 Day 9 6.485 5.76 7.67 Day 11 6.202 5.647.321 Single Day 6 6.691 5.882 7.575 Harvest

Refer FIG.-5 for Log Yield Titers of DEN 2, 3, 4 Virus using 90 rollerbottles (Multiple Harvest vs. Single Harvest)

The cumulative yield of multiple harvests from single batch was muchhigher (around 0.4 to 0.6 log) than yield obtained by single harvest. As0.3 log is equivalent to double of absolute value; this difference ismore significant. Thus, the approach of multiple harvesting is morebeneficial and preferred over single harvest.

EXAMPLE 2

Dengue virus is grown on Vero cells. Thus it is required to removeimpurity from the harvest. Impurities like Host cell DNA is treated withBenzonase.

Effect of Benzonase Concentration and Temperature on Cellular DNAContent and Virus Titer

-   1. Multiple harvests were taken and processed separately to obtain    clarified monovalent virus pools (CMVPs).-   2. These multiple harvests are clarified through 6μ+0.45μ filter and    treated with Benzonase to degrade cellular DNA.-   3. Various experiments were performed to identify the ideal    Benzonase concentration and temperature for treating harvest.

In this experiment Benzonase was added at four different concentration500 units/liter, 1250 units/liter, 2500 units/liter and 5000 units/literat 34±1° C. for 2 hours. It was observed that at 1250, 2500 and 5000units/liter concentration of Benzonase, there was optimum degradation ofDNA. Based on results 1250 units/liter was selected as workingconcentration.

TABLE 7 Effect of Benzonase concentration on DNA content (ng/ml) Control500 U/L 1250 U/L 2500 U/L 5000 U/L Day 5 127 54 5 6 4 Day 7 310 36 7 9 3Day 9 169 19 2 1 5

Refer FIG. 6: Effect of Benzonase concentration on DNA content

Refer FIG. 7: Effect of temperature on Cellular DNA content and virustiter

The dengue virus is sensitive to long time of temperature exposure soshort period of exposure was used in the experiment i.e 2 hours atdifferent temperature. It was observed that there was loss in titer at37° C. while DNA content was low. At 34° C. there was no loss in virustiter and DNA content was low. While at 25° C. and 2 to 8° C. there wasno loss in virus titer as well as DNA degradation was also less. Thus34° C. was selected for process.

EXAMPLE 3 Filtration & Concentration

-   1. Dengue viruses are grown on Vero cells. Thus it is required to    remove impurity from the harvest.-   2. Impurities like Host cell DNA, host cell protein, residual BSA    and residual Benzonase.-   3. Tangential flow filtration (TFF) is a process which we used to    remove these impurities.-   4. Benzonase treated harvest is subjected to TFF for removal of    impurities.

In the initial experiments TFF was done using Millipore V screencassette of 300 KD and Pall T series 300 KD. It was observed that thereis virus loss of ≥3.5 log in permeate. Thus it was planned to change thecassette size to 100 KD. During these experiments it was observed thatin Millipore cassette there was no virus in permeate.

After doing various trials of TFF based on results, 100 KD V screenMerck Millipore cassettes with only 10× concentration process which gaveus desirable product. Virus is concentrated to reduce the requirement ofstorage space for bulk vaccine and removal of impurities. Based on belowtable it is clear that there was no significant loss in virus titer andsignificant removal of Host cell DNA after Benzonase treatment and TFF.

TABLE 8 Host Cell DNA Concentration in ng/ml Intermediate Stage Day ofharvest of Production 5 7 9 DEN 1 CVP 1754.5 2255.9 1631.2 BCVP 186.4298.813 88.201 CMVP 2.238 3.583 4.134 DEN 2 CVP 2430.3 2160.1 2419.2 BCVP215.809 176.026 95.958 CMVP 15.285 8.236 6.409 DEN 3 CVP 1578.6 1340.3522.7 BCVP 93.877 22.9 10.37 CMVP 4.564 4.07 3.43 DEN 4 CVP 1283.1 350.8110.1 BCVP 30.19 53.14 26.81 CMVP 7.22 5.93 8.13 CVP Clarified viruspool (harvest post filtration) BCVP CVP treated with Benzonase CMVPClarified monovalent virus pool (post TFF, addition of stabilizer andpost 0.2 μ filtration)

TABLE 9 Host cell DNA in final product Sr. No. Batch No. DNA (ng/dose) 1Batch 1 0.345 2 Batch 2 0.043 3 Batch 3 0.580

TABLE 10 Mean Virus recovery in 3 consecutive batches (%) Mean SerotypeBatch 1 Batch 2 Batch 3 recovery DEN 1 88.6 92.4 87.1 89.4 DEN 2 99.196.8 94.7 96.9 DEN 3 88.4 94.6 89.2 90.7 DEN 4 98.9 101.4 90.2 96.8

EXAMPLE 4

Study of Various Stabilizers and Optimization of Stabilizer Formulation

Stability of live, attenuated flavivirus immunogenic compositions weretested as a function of potency loss using various stabilizingformulations (e.g., titer loss or Log₁₀PFU/dose).

Dengue monovalent bulks were formulated using different stabilizercombinations as illustrated in table 11 and 12. The principal componentsof these stabilizers were Gelatin, Sorbitol, Sucrose, Glycine,Phosphates (KH₂PO₄, K₂HPO₄), Glutamate, Lactalbumin hydrolysate (LAH)and amino acids as L-Histidine, L-Arginine hydrochloride, L-Alanine,Tricine etc.

TABLE 11 Various Stabilizer Combinations No. Composition of stabilizer AGelatin 2% + Sucrose 20% + Amino acids (2×) B Gelatin 2% + Sucrose 10% +Amino acids (2×) C Gelatin 2% + LAH 0.70% + Sucrose 20% + Amino acids(2×) D Sucrose-Phosphate-Glutamate (2×) + LAH 4% + Glycine 10% + Aminoacids (2×) E Gelatin 12.5% − Sorbitol 25% + Stabilizer- II-Mixingproportion 80:20:10 F Gelatin 12.5% − Sorbitol 25% + Stabilizer-II-Mixing proportion 1:1 G Sucrose 12.5% w/v + Glycine 12.5% w/v (60:40)

Stabilizer-II contains L-Histidine (2.1%), L-Alanine 1%, Tricine (3%),L-Arginine hydrochloride (16%), Lactalbumin hydrolysate (3.5%).

The obtained volume of TFF Viral conc. was stabilized using differentstabilizer combination as described below:

TABLE 12 Final Formulation No. Formulation A  50 ml TFF Conc. + 50 mlStabilizer A (2×) = 100 ml B  50 ml TFF Conc. + 50 ml Stabilizer B (2×)= 100 ml C  50 ml TFF Conc. + 50 ml Stabilizer C (2×) = 100 ml D  50 mlTFF Conc. + 50 ml Stabilizer D (2×) = 100 ml E 320 ml TFF Conc. + 120 mlStabilizer E = 440 ml F  50 ml TFF Conc. + 50 ml Stabilizer F (2×) = 100ml Lyo 1 320 ml TFF Conc. + 120 ml Stabilizer E = 440 ml Lyo 2  50 mlTFF Conc. + 33 ml Stabilizer G = 83 ml

All these formulations were subjected for thermal stability study at 37°C. for 7 days. The samples were tested for infectivity titers by CCID₅₀,intermittently at 0, 1, 3, 5 & 7 days.

The results of infectivity titers of the various liquid formulationsshowed significant drop followed by complete loss (after 1 to 5 days) inrespective dengue virus titers.

This failure of liquid formulation to retain stability prompted toattempt for lyophilized formulations. The dengue monovalent bulkscontaining Gelatin+Sorbitol+Stabilizer II (Stabilizer E) werelyophilized and subjected for thermal stability study at 37° C. for 7days. Samples were tested for infectivity titers by CCID₅₀,intermittently at 0, 1, 3, 5 & 7 days. Results of infectivity titersshowed better stability profile as compared to liquid formulations andsignificantly retained the virus titers. Thus, the approach oflyophilization of dengue monovalent formulation successfully overcamethe problem of poor stability & found significant reduction of loss invirus titers.

Also another StabilizerG comprising of Sucrose+Glycine was tried out;and was excellent in form of lyophilized formulation for all four dengueviruses. Samples were tested for infectivity titers by CCID₅₀,intermittently at 0, 1, 3, 5 & 7 days. Results of infectivity titersshowed better stability profile as compared to other stabilizers e.g.Gelatin+Sorbitol+stabilizer II. Also, it is easy to prepare and use;since it is sourced from non-animal origin.

TABLE 13 DEN 1 Titer CCID₅₀/ml (Liquid vs. Lyophilized) Storage time A BC D E F LYO 1 LYO 2 0 7.21 7.35 7.07 7.07 7.21 7.21 5.78 5.62 1 5.214.07 4.07 4.78 2.78 2.78 5.78 5.59 3 2.92 2.78 0 2.64 2.64 2.64 5.355.46 5 0 0 0 0 0 0 4.78 5.31 7 — — — — — — 4.64 5.18

Refer FIG. 8: DEN-1 titer CCID₅₀/ml (Liquid vs. Lyophilized)

TABLE 14 DEN 2 Titer CCID₅₀/ml (Liquid vs. Lyophilized) Storage time A BC D LYO 1 LYO 2 0 5.625 5.75 4.88 5 4.64 4.7 1 0 0 0 0 4.21 4.52 3 — — —— 4.07 4.43 5 — — — — 3.75 4.26 7 — — — — 3.5 4.15

Refer FIG. 9: DEN 2 Titer CCID₅₀/ml (Liquid vs. Lyophilized)

TABLE 15 DEN 3 Titer CCID₅₀/ml (Liquid vs. Lyophilized) Storage time A BC D F LYO 1 LYO 2 0 6.92 7.07 6.35 6.92 6.78 6.83 6.76 1 4.21 4.92 3.64.07 0 6.69 6.65 3 0 0 0 0 NT 6.58 6.56 5 NT NT NT NT NT 6.43 6.44 7 NTNT NT NT NT 6.35 6.33 NT- Not tested

Refer FIG. 10: DEN-3 Titer CCID₅₀/ml (Liquid vs. Lyophilized)

TABLE 16 DEN 4 Titer CCID₅₀/ml (Liquid vs. Lyophilized) Storage time LYO1 LYO 2 0 4.64 4.78 1 4.64 4.75 3 4.64 4.74 5 4.25 4.57 7 3.87 4.32

Refer FIG. 11: DEN 4 Titer CCID₅₀/ml (Lyophilized)

With reference to all above results of stability study, the betterstability profile was obtained with LYO 2 (Sucrose & Glycine). Hence,the stabilizer composition of Sucrose & Glycine was further optimized toget more stable formulation.

EXAMPLE 5

Lyophilization Conditions

For dengue vaccine development initially we have tried various differentstabilizers formulations for liquid vaccine, but it was found that virusdoes not remain stable in liquid formulation and there was significanttiter loss with liquid formulation within 5 days stored at 37° C. Liquidformulation doesn't work for dengue vaccine. So we further decided tocarry out next trials of dengue vaccine bulk using lyophilisation.

Different lyophilisation trials were planned and carried out on Denguemonovalent as well as tetravalent bulk to study suitability ofstabilizer for vaccine formulation and stability of all four virusserotypes in tetravalent mixture during storage at low temperature.

Our study trials showed that virus remains more stable in lyophilizedform than in liquid form and no virus titer loss was observed afterlyophilisation as illustrated in example 4.

We opted for lyophilized form dengue vaccine to obtain better stabilityof the product. Lyophilisation trials were carried out by using DengueCMVP-bulk containing Gelatin-Sorbitol or Sucrose-Glycine as stabilizersas illustrated in example 4. As gelatin is of porcine origin andnowadays there are some ethical issues for its use in vaccine. Alsocomplexity in its preparation which requires hydrolysis of gelatin athigh temperature. With use of Gelatin-Sorbitol stabilizer no consistencyin infectivity titers of all four serotypes and considerable virus losswas observed from thawing to lyophilisation step.

After some lyophilisation trials of dengue bulk withGelatin+sorbitol+stabilizer II we shifted to Sucrose+Glycine asillustrated in example 4 and the lyophilisation trials showed bettervirus stability for LYO 2 formulation as compared to LYO 1 formulation.

TABLE 17 Lyophilization Cycle Optimization Moisture Trial Lyo. cycle inTitre Loss content No. Hours. (pfu/ml) (w/v %) 1 43 Hrs.   0.056 2.761 242 Hrs.   0.089 3.280 3 27 Hrs. +0.017 3.343 4 35 Hrs.   0.292 3.098 539 Hrs.   0.154 2.575 6 45 Hrs.   0.282 2.814 7 56 Hrs.   0    2.495

In Trial No.1 to 7 lyophilization cycle duration was changed fromstandard cycle of 56 Hrs. The reduction in the hours of lyophilizationcycle had an effect on titer loss and moisture content. There was nomajor loss in virus titer however; moisture content increased from 2.50%to 3.34%w/w as there was reduction in the cycle time. Hence, thelyophilization cycle duration was set around 56 Hrs.

TABLE 18 Lyophilization process parameters Activity Parameters Freezing−45° C. for 13.5 ± 2 hrs Condenser cooling ≤1 hrs Chamber Evacuation≤1.30 hrs Primary drying −45° C. to 25° C. for 33 ± 3 hrs Secondarydrying 25° C. for 8 ± 0.5 hrs

Refer FIG. 12: Lyophilization Cycle

EXAMPLE 6 Stability Data Post Lyophilization of Dengue Virus MonovalentBulk at 37±1° C. for 14 days

Dengue virus monovalent bulks (DEN 1,2,3,4) were formulated with varyingconcentrations of sucrose and glycine to obtain the final formulationcomprising concentration of sucrose and glycine as enclosed in table 19(SG1, SG2, SG3, SG4, SG5, SG6). The stabilized monovalent formulationswere lyophilized using standard lyophilization protocol. All lyophilizedmonovalent and tetravalent dengue virus formulations as enclosed intable below were subjected to thermal stability study at 37±1° C. for 14days. The samples were collected at respective time points and testedfor infectivity titers.

TABLE 19 Sucrose + Glycine Formulations Formulation code Sucrose GlycineSG1   5% w/v 3% w/v SG2   3% w/v 5% w/v SG3   5% w/v 5% w/v SG4  10% w/v7% w/v SG5 4.5% w/v 5% w/v SG6   6% w/v 6% w/v

TABLE 20A Dengue Monovalent Vaccine Composition SG1 Quantity perComponent dose of 0.5 ml Dengue virus serotype 1 NLT log₁₀ 2.5 PFU (rDEN1Δ30) Sucrose 5% w/v Glycine 3% w/v

TABLE 20B Dengue Monovalent Vaccine Composition SG2 Quantity perComponent dose of 0.5 ml Dengue virus serotype 1 NLT log₁₀ 2.5 PFU (rDEN1Δ30) Sucrose 3% w/v Glycine 5% w/v

TABLE 20C Dengue Monovalent Vaccine Composition SG3 Quantity perComponent dose of 0.5 ml Dengue virus serotype 1 NLT log₁₀ 2.5 PFU (rDEN1Δ30) Sucrose 5% w/v Glycine 5% w/v

TABLE 20D Dengue Monovalent Vaccine Composition SG4 Quantity perComponent dose of 0.5 ml Dengue virus serotype 1 NLT log₁₀ 2.5 PFU (rDEN1Δ30) Sucrose 10% w/v Glycine  7% w/v

TABLE 20E Dengue Monovalent Vaccine Composition SG5 Quantity perComponent dose of 0.5 ml Dengue virus serotype 1 NLT log₁₀ 2.5 PFU (rDEN1Δ30) Sucrose 4.5% w/v Glycine   5% w/v

TABLE 20F Dengue Monovalent Vaccine Composition SG6 Quantity perComponent dose of 0.5 ml Dengue virus serotype 1 (rDEN 1Δ30) NLT log₁₀2.5 PFU Sucrose 6% w/v Glycine 6% w/v

TABLE 21A Dengue Monovalent Vaccine Composition SG1 Quantity perComponent dose of 0.5 ml Dengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀3.0 PFU Sucrose 5% w/v Glycine 3% w/v

TABLE 21B Dengue Monovalent Vaccine Composition SG2 Quantity perComponent dose of 0.5 ml Dengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀3.0 PFU Sucrose 3% w/v Glycine 5% w/v

TABLE 21C Dengue Monovalent Vaccine Composition SG3 Quantity perComponent dose of 0.5 ml Dengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀3.0 PFU Sucrose 5% w/v Glycine 5% w/v

TABLE 21D Dengue Monovalent Vaccine Composition SG4 Quantity perComponent dose of 0.5 ml Dengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀3.0 PFU Sucrose 10% w/v Glycine  7% w/v

TABLE 21E Dengue Monovalent Vaccine Composition SG5 Quantity perComponent dose of 0.5 ml Dengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀3.0 PFU Sucrose 4.5% w/v Glycine   5% w/v

TABLE 21F Dengue Monovalent Vaccine Composition SG6 Quantity perComponent dose of 0.5 ml Dengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀3.0 PFU Sucrose 6% w/v Glycine 6% w/v

TABLE 22A Dengue Monovalent Vaccine Composition SG1 Quantity perComponent dose of 0.5 ml Dengue virus serotype 3 (rDEN 3Δ30/31) NLTlog₁₀ 2.5 PFU Sucrose 5% w/v Glycine 3% w/v

TABLE 22B Dengue Monovalent Vaccine Composition SG2 Quantity perComponent dose of 0.5 ml Dengue virus serotype 3 (rDEN 3Δ30/31) NLTlog₁₀ 2.5 PFU Sucrose 3% w/v Glycine 5% w/v

TABLE 22C Dengue Monovalent Vaccine Composition SG3 Quantity perComponent dose of 0.5 ml Dengue virus serotype 3 (rDEN 3Δ30/31) NLTlog₁₀ 2.5 PFU Sucrose 5% w/v Glycine 5% w/v

TABLE 22D Dengue Monovalent Vaccine Composition SG4 Quantity perComponent dose of 0.5 ml Dengue virus serotype 3 (rDEN 3Δ30/31) NLTlog₁₀ 2.5 PFU Sucrose 10% w/v Glycine  7% w/v

TABLE 22E Dengue Monovalent Vaccine Composition SG5 Quantity perComponent dose of 0.5 ml Dengue virus serotype 3 (rDEN 3Δ30/31) NLTlog₁₀ 2.5 PFU Sucrose 4.5% w/v Glycine   5% w/v

TABLE 22F Dengue Monovalent Vaccine Composition SG6 Quantity perComponent dose of 0.5 ml Dengue virus serotype 3 (rDEN 3Δ30/31) NLTlog₁₀ 2.5 PFU Sucrose 6% w/v Glycine 6% w/v

TABLE 23A Dengue Monovalent Vaccine Composition SG1 Quantity perComponent dose of 0.5 ml Dengue virus serotype 4 (rDEN 4Δ30) NLT log₁₀2.5 PFU Sucrose 5% w/v Glycine 3% w/v

TABLE 23B Dengue Monovalent Vaccine Composition SG2 Quantity perComponent dose of 0.5 ml Dengue virus serotype 4 (rDEN 4Δ30) NLT log₁₀2.5 PFU Sucrose 3% w/v Glycine 5% w/v

TABLE 23C Dengue Monovalent Vaccine Composition SG3 Quantity per dose ofComponent 0.5 ml Dengue virus serotype 4 (rDEN 4Δ30) NLT log₁₀ 2.5 PFUSucrose 5% w/v Glycine 5% w/v

TABLE 23D Dengue Monovalent Vaccine Composition SG4 Quantity per dose ofComponent 0.5 ml Dengue virus serotype 4 (rDEN 4Δ30) NLT log₁₀ 2.5 PFUSucrose 10% w/v Glycine  7% w/v

TABLE 23E Dengue Monovalent Vaccine Composition SG5 Quantity per dose ofComponent 0.5 ml Dengue virus serotype 4 (rDEN 4Δ30) NLT log₁₀ 2.5 PFUSucrose 4.5% w/v Glycine   5% w/v

TABLE 23F Dengue Monovalent Vaccine Composition SG6 Quantity per dose ofComponent 0.5 ml Dengue virus serotype 4 (rDEN 4Δ30) NLT log₁₀ 2.5 PFUSucrose 6% w/v Glycine 6% w/v

TABLE 24 Dengue Tetravalent Vaccine Composition SG1 Quantity per dose ofComponent 0.5 ml Dengue virus serotype 1 (rDEN 1Δ30) NLT log₁₀ 2.5 PFUDengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀ 3.0 PFU Dengue virusserotype 3 (rDEN 3Δ30/31) NLT log₁₀ 2.5 PFU Dengue virus serotype 4(rDEN 4Δ30) NLT log₁₀ 2.5 PFU Sucrose 5% w/v Glycine 3% w/v

TABLE 25 Dengue Tetravalent Vaccine Composition SG2 Quantity per dose ofComponent 0.5 ml Dengue virus serotype 1 (rDEN 1Δ30) NLT log₁₀ 2.5 PFUDengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀ 3.0 PFU Dengue virusserotype 3 (rDEN 3Δ30/31) NLT log₁₀ 2.5 PFU Dengue virus serotype 4(rDEN 4Δ30) NLT log₁₀ 2.5 PFU Sucrose 3% w/v Glycine 5% w/v

TABLE 26 Dengue Tetravalent Vaccine Composition SG3 Quantity per dose ofComponent 0.5 ml Dengue virus serotype 1 (rDEN 1Δ30) NLT log₁₀ 2.5 PFUDengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀ 3.0 PFU Dengue virusserotype 3 (rDEN 3Δ30/31) NLT log₁₀ 2.5 PFU Dengue virus serotype 4(rDEN 4Δ30) NLT log₁₀ 2.5 PFU Sucrose 5% w/v Glycine 5% w/v

TABLE 27 Dengue Tetravalent Vaccine Composition SG4 Quantity per dose ofComponent 0.5 ml Dengue virus serotype 1 (rDEN 1Δ30) NLT log₁₀ 2.5 PFUDengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀ 3.0 PFU Dengue virusserotype 3 (rDEN 3Δ30/31) NLT log₁₀ 2.5 PFU Dengue virus serotype 4(rDEN 4Δ30) NLT log₁₀ 2.5 PFU Sucrose 10% w/v Glycine  7% w/v

TABLE 28 Dengue Tetravalent Vaccine Composition SG5 Quantity per dose ofComponent 0.5 ml Dengue virus serotype 1 (rDEN 1Δ30) NLT log₁₀ 2.5 PFUDengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀ 3.0 PFU Dengue virusserotype 3 (rDEN 3Δ30/31) NLT log₁₀ 2.5 PFU Dengue virus serotype 4(rDEN 4Δ30) NLT log₁₀ 2.5 PFU Sucrose 4.5% w/v Glycine   5% w/v

TABLE 29 Dengue Tetravalent Vaccine Composition SG6 Quantity per dose ofComponent 0.5 ml Dengue virus serotype 1 (rDEN 1Δ30) NLT log₁₀ 2.5 PFUDengue virus serotype 2 (rDEN 2/4Δ30) NLT log₁₀ 3.0 PFU Dengue virusserotype 3 (rDEN 3Δ30/31) NLT log₁₀ 2.5 PFU Dengue virus serotype 4(rDEN 4Δ30) NLT log₁₀ 2.5 PFU Sucrose 6% w/v Glycine 6% w/v

TABLE 30 DEN 1 Titer Log₁₀ pfu/ml post lyophilization Storage time SG1SG2 SG3 SG4 SG5 SG6  0 7.105 7.024 7.217 7.185 7.060 6.994  1 7.0207.051 7.300 7.242 7.021 6.950  3 6.898 6.917 7.152 7.082 6.935 6.886  56.925 6.720 6.954 6.981 6.724 6.817  7 6.700 6.510 6.872 6.732 6.6006.746 14 6.346 6.180 6.585 6.500 6.312 6.395

Refer FIG. 13: DEN-1 titer Log₁₀ pfu/ml post lyophilization

TABLE 31 DEN 2 Titer Log₁₀ pfu/ml post lyophilization Storage time SG1SG2 SG3 SG4 SG5 SG6  0 6.775 6.821 6.824 6.780 6.900 6.772  1 6.6506.760 6.797 6.802 6.855 6.780  3 6.684 6.617 6.712 6.689 6.723 6.665  56.521 6.588 6.610 6.584 6.692 6.610  7 6.246 6.405 6.482 6.410 6.5366.538 14 6.060 6.144 6.227 6.197 6.272 6.294

Refer FIG. 14: DEN-2 titer Log₁₀ pfu/ml post lyophilization

TABLE 32 DEN 3 Titer Log₁₀ pfu/ml post lyophilization Storage time SG1SG2 SG3 SG4 SG5 SG6  0 5.929 5.864 5.882 5.877 5.765 5.800  1 5.7885.712 5.800 5.762 5.692 5.728  3 5.621 5.644 5.725 5.688 5.566 5.610  55.546 5.571 5.622 5.621 5.487 5.543  7 5.440 5.473 5.496 5.492 5.3515.414 14 5.132 5.082 5.184 5.131 5.139 5.220

Refer FIG. 15: DEN-3 titer Log₁₀ pfu/ml post lyophilization

TABLE 33 DEN 4 Titer Log₁₀ pfu/ml post lyophilization Storage time SG1SG2 SG3 SG4 SG5 SG6  0 7.429 7.441 7.512 7.464 7.500 7.471  1 7.3667.400 7.504 7.471 7.432 7.388  3 7.302 7.343 7.438 7.402 7.386 7.295  57.210 7.219 7.393 7.330 7.277 7.197  7 7.157 7.134 7.268 7.225 7.1507.065 14 6.809 6.792 7.021 6.96 6.880 6.782

Refer FIG. 16: DEN-4 titer Log₁₀ pfu/ml post lyophilization

Aforementioned thermal stability results for lyophilized monovalentformulations containing varying concentrations of sucrose and glycinestabilizers; indicate that the concentration of both sucrose & glycineplays major role in maintaining the virus infectivity titer.

Comparatively more variation in virus titers was observed with SG1 & SG2formulations. No significant difference was observed with SG3, SG4, SG5, SG 6, formulations in all four serotypes. However, SG3 i.e. 5%Sucrose & 5% Glycine formulation was the choice of stabilizercomposition for further batches.

EXAMPLE 7

Stability Data of Dengue Tetravalent Vaccine, Live Attenuated(Recombinant, Lyophilized)

Dengue Tetravalent Vaccine (DTV) (live, attenuated, Recombinant) havinga combination of serotypes (DEN-1, DEN 2, DEN-3, DEN-4) stabilized usingsucrose-glycine (SG) composition as enclosed in Example 6 and furtherlyophilized according to example 5 in 3 ml tubular USP type-1 glassvials. Container closure system consists of bromobutyl rubber stoppersand flip-off aluminium and plastic caps seals.

The stability and quality of SG-stabilized vaccine was evaluated in theabove-said container closure system for following studies in line withICH requirement to support the expiry period of DP.

Stability indicating parameters for long-term/real time stabilitystudies were following:

1. Virus titers of each serotype

2. pH

3. Moisture content

1. Dengue Tetravalent Vaccine Stabilitydataat 2-8° C. upto 12 monthspost lyophilization:

TABLE 34A DEN-1 titer Log₁₀ pfu/0.5 ml data post lyophilization at 2- 8°C. up to 12 months No. 0 1 2 3 6 9 12 Batch4 4.62 4.43 4.29 4.44 4.614.4 4.65 Batch5 5.17 4.8 4.69 4.85 4.83 4.67 4.59 Batch6 4.88 4.57 4.574.7 4.55 4.62 4.59

Refer FIG. 17A: DEN-1 titer Log₁₀ pfu/0.5 ml data post lyophilization at2-8° C. upto 12 months

TABLE 34B DEN-2 titer Log₁₀ pfu/0.5 ml data post lyophilization at 2- 8°C. up to 12 months No. 0 1 2 3 6 9 12 Batch4 5.22 5.06 4.93 4.92 5.144.85 4.88 Batch5 5.03 4.79 4.82 4.81 4.80 4.78 4.38 Batch6 5.01 4.934.85 4.86 4.88 4.63 4.24

Refer FIG. 17B: DEN-2 titer Log₁₀ pfu/0.5 ml data post lyophilization at2-8° C. upto 12 months

TABLE 34C DEN-3 titer Log₁₀ pfu/0.5 ml data post lyophilization at 2- 8°C. up to 12 months No. 0 1 2 3 6 9 12 Batch4 4.62 4.31 4.25 4.38 4.564.33 4.52 Batch5 5.30 4.85 4.51 5.05 4.96 4.69 4.45 Batch6 5.00 4.574.52 4.69 4.59 4.54 4.05

Refer FIG. 17C: DEN-3 titer Log₁₀ pfu/0.5 ml data post lyophilization at2-8° C. upto 12 months

TABLE 34D DEN-4 titer Log₁₀ pfu/0.5 ml data post lyophilization at 2- 8°C. up to 12 months No. 0 1 2 3 6 9 12 Batch4 5.28 5.09 4.89 4.92 5.064.91 4.90 Batch5 5.27 5.02 4.93 4.9 5.04 4.84 4.38 Batch6 4.78 4.34 4.534.49 4.66 4.36 3.97

Refer FIG. 17D: DEN-4 titer Log₁₀ pfu/0.5 ml data post lyophilization at2-80C. upto 12 months

pH and moisture content was estimated at 2-8° C. upto 12 months. pHremained within the range of 7.6 to 7.8 (FIG. 18). Residual moisturecontent remained below than 3.0% w/w up to 12 months storage, (FIG. 19).

2. Dengue Tetravalent Vaccine Stability data at 25° C.±2° C. and 60%±5%relative humidity upto 6 months post lyophilization:

TABLE 35A DEN-1 titer Log₁₀ pfu/0.5 ml data post lyophilization at 25°C. up to 6 months No. 0 1 2 3 6 Batch4 4.32 4.19 4.44 4.47 4.39 Batch54.87 4.82 4.87 4.81 4.74 Batch6 4.57 4.70 4.77 4.62 4.61

Refer FIG. 20A: DEN-1 titer Log₁₀ pfu/0.5 ml data post lyophilization at25° C.±2° C. upto 6 months

TABLE 35B DEN-2 titer Log₁₀ pfu/0.5 ml data post lyophilization at 25°C. upto 6 months No. 0 1 2 3 6 Batch4 4.92 4.95 5.11 5.10 4.87 Batch54.73 4.69 4.89 4.91 4.74 Batch6 4.71 4.72 4.85 4.81 4.67

Refer FIG. 20B: DEN-2 titer Log₁₀ pfu/0.5 ml data post lyophilization at25° C.±2° C. upto 6 months

TABLE 35C DEN-3 titer Log₁₀ pfu/0.5 ml data post lyophilization at 25°C. upto 6 months No. 0 1 2 3 6 Batch4 4.32 4.36 4.35 4.37 4.40 Batch54.99 4.86 4.91 5.03 4.85 Batch6 4.69 4.66 4.76 4.84 4.65

Refer FIG. 20C: DEN-3 titer Log₁₀ pfu/0.5 ml data post lyophilization at25° C.±2° C. upto 6 months

TABLE 35D DEN-4 titer Log₁₀ pfu/0.5 ml data post lyophilization at 25°C. upto 6 months No. 0 1 2 3 6 Batch4 4.98 4.95 5.11 5.08 4.91 Batch54.97 4.86 5.08 5.03 4.89 Batch6 4.48 4.43 4.57 4.39 4.36

Refer FIG. 20D: DEN-4 titer Log₁₀ pfu/0.5 ml data post lyophilization at25° C.±2° C. upto 6 months

The pH and moisture content was estimated on initial and final timepoint at 25° C.±2° C. upto 6 months(6 months post exposure). No changein pH occurred on storage at accelerated conditions for 6 monthscompared to initial values (p<0.001); Mean±3SD shifted from 7.60-7.79 to7.54-7.71 (FIG. 21). Residual moisture content remained within upperlimit of 3% w/w, Mean±3SD of 1.845-2.774% w/w after 6 months poststorage (FIG. 22).

3. Dengue Tetravalent Vaccine Stability data at 37° C.±1° C. upto 7 dayspost lyophilization:

Batches were exposed to 37° C.±1° C. for 7 days. Virus serotypes(DEN1-4) were titrated and loss in titers was calculated. Loss in titerswas consistent in all batches. Average Log₁₀ loss in virus titers andstandard deviation of Dengue 1 to Dengue 4 serotypes in lyophilized DTVwere 0.604±0.117, 0.607±0.066, 0.548±0.130, 0.684±0.109 respectively(FIG. 23).

TABLE 36 Dengue Tetravalent Vaccine Stability data Post lyophilizationat 37° C. ± 1° C. upto 7 days B. No. DEN1 DEN2 DEN3 DEN4 Batch1 0.600.68 0.60 0.68 Batch2 0.68 0.61 0.69 0.62 Batch3 0.50 0.54 0.41 0.61Batch4 0.67 0.67 0.52 0.76 Batch5 0.69 0.50 0.65 0.69 Batch6 0.71 0.650.63 0.87

Refer FIG. 23: Dengue Tetravalent Vaccine Stability data at 37° C.±1° C.upto 7 days post lyophilization

4. Dengue Tetravalent Vaccine Stability data at 42° C.±1° C. upto 7 dayspost lyophilization:

Stability of Dengue Tetravalent Vaccine (DTV) (live, attenuated) wasevaluated on a representative batch. Lyophilized finished product vialswere exposed to thermal stress condition at 42° C. for 7 days. Eachvirus serotype (DEN1-4) was titrated at the end of exposure period,compared with the initial titer and loss in titers was calculated.

(Refer FIG. 24)

TABLE 37 Dengue Tetravalent Vaccine Stability data Post lyophilizationat 42° C. ± 1° C. upto 7 days Days post Log₁₀ loss in virus titers ofDTV (pfu/0.5 ml) exposure DEN1 DEN2 DEN3 DEN4 0 day 4.41 4.43 4.26 4.067 day 3.91 3.81 3.83 3.42 Loss in titer 0.50 0.62 0.43 0.64

5. Dengue Tetravalent Vaccine Stability data at 55° C.±1° C. upto 2 dayspost lyophilization:

Stability of Dengue Tetravalent Vaccine (DTV) (live, attenuated) wasevaluated on a representative batch. Lyophilized finished product vialswere exposed to thermal stress condition at 55° C. for 2 days. Eachvirus serotype (DEN1-4) was titrated at the end of exposure period,compared with the initial titer and loss in titers was calculated.

While the present invention has been described with respect to certainpreferred embodiments, it will be apparent to those skilled in the artthat various changes and modifications may be made without departingfrom the scope of the invention as defined in the following claims.

1. An immunogenic composition comprising: a) one or more live attenuatedflaviviruses; b) one or more carbohydrates about 3 to 6% (w/v); c) oneor more amino acids about 3 to 6% (w/v); wherein, the composition isamenable to freeze-drying treatments and the reconstituted compositionpreserves the desired characteristics of a virus, including virusviability, immunogenicity and stability.
 2. The immunogenic compositionas claimed in claim 1, wherein the one or more live attenuatedflaviviruses is recombinant flaviviruses or chimeric flaviviruses. 3.The immunogenic composition as claimed in claim 1, wherein the one ormore live attenuated flaviviruses is selected from a group consisting ofdengue (DEN) virus, yellow fever (YF) virus, Japanese encephalitis (JE)virus, Kunjin virus, West Nile (WN) virus, tick-borne encephalitis (TBE)virus, St. Louis encephalitis virus, Murray Valley encephalitis virus,Zika virus, a chimeric dengue virus (yellow fever-dengue) virus, achimeric YF-WN (yellow fever-West Nile virus) virus, a chimeric YF-JE(yellow fever-Japanese encephalitis) virus, and any related flavivirusthereof.
 4. The immunogenic composition as claimed in claim 1, whereinthe one or more live attenuated flaviviruses comprises dengue (DEN)virus, and optionally comprises a plurality of live attenuated dengue(DEN) viruses of different serotypes selected from a group consisting ofDEN-1, DEN-2, DEN-3 and DEN-4.
 5. The immunogenic composition as claimedin claim 4, wherein the live attenuated dengue (DEN) virus istetravalent comprising dengue virus serotypes DEN-1, DEN-2, DEN-3 andDEN-4.
 6. The immunogenic composition as claimed in claim 5, wherein thelive attenuated dengue (DEN) virus is recombinant dengue viruses and/ora chimeric dengue viruses comprising a first nucleotide sequenceencoding at least one structural proteins from a first dengue virus anda second nucleotide sequence encoding non-structural proteins from asecond dengue virus.
 7. The immunogenic composition as claimed in claim6, wherein said live attenuated dengue virus serotypes DEN-1, DEN-2,DEN-3 and DEN-4 carry 30 nucleotide deletion denoted Δ 30 mutationand/or carry 31 nucleotide deletion denoted Δ 31 mutation in the 3′untranslated region of dengue virus genome.
 8. The immunogeniccomposition as claimed in claim 7, wherein said live attenuated denguevirus serotypes DEN-1, DEN-2, DEN-3 and DEN-4 have a phenotype which istemperature sensitive in Vero cells or a human liver cell line HuH-7. 9.The immunogenic composition as claimed in claim 1, wherein the one ormore carbohydrates is selected from the group consisting of naturalcarbohydrates, synthetic carbohydrates, monosaccharides, disaccharides,trisaccharides, oligosaccharides, reducing sugars, non-reducing sugars,sugar alcohols, polyols, polyhydroxyl compounds, chemically modifiedcarbohydrates, sugar copolymers, and glass transition facilitatingagents wherein the glass transition facilitating agents is selected fromthe group consisting of sucrose, mannitol, trehalose, mannose,raffinose, lactitol, lactobionic acid, glucose, maltulose,iso-maltulose, maltose, lactose sorbitol, dextrose, fucose and acombination thereof.
 10. The immunogenic composition as claimed in claim9, wherein at least one of the carbohydrates is sucrose.
 11. Theimmunogenic composition as claimed in claim 1, wherein the one or moreamino acids is selected from the group consisting of leucine,iso-leucine, histidine, glycine, glutamine, arginine, lysine, alanineand a combination thereof.
 12. The immunogenic composition as claimed inclaim 11, wherein at least one of the amino acids is glycine.
 13. Theimmunogenic composition as claimed in claim 1, further comprising anadjuvant.
 14. The immunogenic composition as claimed in claim 13,wherein the adjuvant comprises an aluminum salt, and optionally at leastone of aluminum hydroxide, aluminum phosphate, aluminumhydroxyphosphate, and potassium aluminum sulfate.
 15. The immunogeniccomposition as claimed in claim 1, further comprising at least oneadditional immunostimulatory component selected from the groupconsisting of: an oil and water emulsion, MF-59, a liposome, alipopolysaccharide, a saponin, lipid A, lipid A derivatives,Monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, AS01, AS03,an oligonucleotide, an oligonucleotide comprising at least oneunmethylated CpG and/or a liposome, Freund's adjuvant, Freund's completeadjuvant, Freund's incomplete adjuvant, polymers, co-polymers such aspolyoxyethylene-polyoxypropylene copolymers, including blockco-polymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, TLR-4agonists, flagellin, flagellins derived from gram negative bacteria,TLR-5 agonists, fragments of flagellins capable of binding to TLR-5receptors, QS-21, ISCOMS, and saponin combination with sterols andlipids.
 16. The immunogenic composition as claimed in claim 1, whereinthe composition is lyophilized (freeze-dried).
 17. The immunogeniccomposition as claimed in claim 16, wherein the lyophilized compositionis reconstituted with an aqueous solution selected from saline and WFI(water for injection), and wherein the final pH of the reconstitutedimmunogenic composition is 7-8.
 18. (canceled)
 19. The immunogeniccomposition as claimed in claim 8, wherein the dengue virus ispropagated in vero cell line ATCC CCL-81 (cGMPVero, Kidney African GreenMonkey—Cercopithecus aeothiops; available from the ATCC, Manassas, Va.,USA).
 20. (canceled)
 21. The immunogenic composition as claimed in claim4, wherein the dengue virus is present at a dose of not less than 2.5log₁₀ PFU per 0.5 ml.
 22. An immunogenic composition comprising: a)Dengue virus serotype 1 (rDEN 1Δ30), b) Dengue virus serotype 2 (rDEN2/4Δ30), c) Dengue virus serotype 3 (rDEN 3Δ30/31), d) Dengue virusserotype 4 (rDEN 4Δ30), e) Sucrose about 3 to 6% (w/v), and f) Glycineabout 3 to 6% (w/v), wherein, the composition is amenable tofreeze-drying treatments and a reconstituted composition preserves thedesired characteristics of a virus, including virus viability,immunogenicity and stability.
 23. The immunogenic composition as claimedin claim 22 comprising: a) Dengue virus serotype 1 (rDEN 1Δ30), b)Dengue virus serotype 2 (rDEN 2/4Δ30), c) Dengue virus serotype 3 (rDEN3Δ30/31), d) Dengue virus serotype 4 (rDEN 4Δ30), e) Sucrose about 4 to5% (w/v), and f) Glycine about 4 to 5% (w/v), wherein, the compositionis amenable to freeze-drying treatments and a reconstituted compositionpreserves the desired characteristics of a virus, including virusviability, immunogenicity and stability.
 24. The immunogenic compositionas claimed in claim 22 comprising: a) Dengue virus serotype 1 (rDEN1Δ30), NLT 2.5 log₁₀ PFU per 0.5 ml, b) Dengue virus serotype 2 (rDEN2/4Δ30), NLT 3.0 log₁₀ PFU per 0.5 ml, c) Dengue virus serotype3 (rDEN3Δ30/31), NLT 2.5 log₁₀ PFU per 0.5 ml, d) Dengue virus serotype 4 (rDEN4Δ30), NLT 2.5 log₁₀ PFU per 0.5 ml, e) Sucrose about 5% (w/v), and f)Glycine about 5% (w/v), wherein, the composition is amenable tofreeze-drying treatments and a reconstituted composition preserves thedesired characteristics of a virus, including virus viability,immunogenicity and stability.
 25. The immunogenic composition as claimedin claim 22 comprising: a) Dengue virus serotype 1 (rDEN 1Δ30), NLT 2.5log₁₀ PFU per 0.5 ml, b) Dengue virus serotype 2 (rDEN 2/4Δ30), NLT 3.0log₁₀ PFU per 0.5 ml, c) Dengue virus serotype3 (rDEN 3Δ30/31), NLT 2.5log₁₀ PFU per 0.5 ml, d) Dengue virus serotype 4 (rDEN 4Δ30), NLT 2.5log₁₀ PFU per 0.5 ml, e) Sucrose about 4.5% (w/v), and f) Glycine about5% (w/v), wherein, the composition is amenable to freeze-dryingtreatments and a reconstituted composition preserves the desiredcharacteristics of a virus, including virus viability, immunogenicityand stability.
 26. The immunogenic composition as claimed in claim 22comprising: a) Dengue virus serotype 1 (rDEN 1Δ30), NLT 2.5 log₁₀ PFUper 0.5 ml, b) Dengue virus serotype 2 (rDEN 2/4Δ30), NLT 3.0 log₁₀ PFUper 0.5 ml, c) Dengue virus serotype3 (rDEN 3Δ30/31), NLT 2.5 log₁₀ PFUper 0.5 ml, d) Dengue virus serotype 4 (rDEN 4Δ30), NLT 2.5 log₁₀ PFUper 0.5 ml, e) Sucrose about 6% (w/v), and f) Glycine about 6% (w/v),wherein, the composition is amenable to freeze-drying treatments and areconstituted composition preserves the desired characteristics of avirus, including virus viability, immunogenicity and stability.
 27. Theimmunogenic composition as claimed in claim 17, wherein the aqueoussolution used for reconstitution of lyophilized immunogenic compositionadditionally comprises of a preservative selected from the group of2-phenoxyethanol, Benzethonium chloride (Phemerol), Phenol, Thiomersal,Formaldehyde, methyl and propyl parabens, benzyl alcohol andcombinations thereof.
 28. (canceled)
 29. A method of manufacturing animmunogenic composition, comprising: a. one or more live attenuateddengue (DEN) viruses; b. sucrose 3 to 6% (w/v); and c. glycine 3 to 6%(w/v); said method comprising the following steps: a) MultipleHarvesting of Supernatant comprising at least one serotype of denguevirus in MEM with Hanks salt solution additionally containing Dextrose,L-Glutamine and Sodium Bicarbonate; b) Filtering the viral harvest bydirect flow filtration (DFF) through at least one clarification filterhaving a pore size of between about 6 micrometers to 0.45 micrometers;c) Treating the viral harvest with a Benzonase having a concentration inthe range of 0.5 units/ml to 5 units/ml at 34±1° C. for at least 2hours; d) Concentrating the viral harvest by tangential flow filtration(TFF) using membrane with a molecular weight cut off (MWCO) of 100 kDa;e) Stabilizing the viral harvest with a stabilizing agent comprisingsucroseat at a concentration of 3 to 6% (w/v) and glycine at aconcentration of 3 to 6% (w/v) to form a stabilized viral harvest; f)Clarification of Sterilizing the stabilized viral harvest by DFF throughat least one clarification filter having a pore size of between about0.8 micrometers to 0.2 micrometers to form a sterilized viral harvest ofpurified virus, wherein the overall recovery of purified viruses is atleast 50%; and g) Optionally freeze drying the sterilized viral harvestcomprising the step of freezing, primary drying and secondary drying,wherein a. the freezing step comprises freezing at −45° C. for 690minutes to 930 minutes, b. the primary drying step comprises ramping at+0.5° C./minute to 0° C./minute to achieve a shelf temperature of −25°C., holding for 1800 minutes to 1980 minutes, and c. the secondarydrying step comprises ramping at +0.5° C./minute to 1.0° C./minute toachieve a shelf temperature of +25° C., holding for 420 minutes to 540minutes. 30.-31. (canceled)
 32. The method as claimed in claim 29,wherein the viral harvest is treated with benzonase having aconcentration 1.25 units/ml.
 33. The method as claimed in claim 29,wherein the viral harvest is subjected to tangential flow filtration(TFF) resulting in at least 10× concentration of the viral harvest.34.-42. (canceled)
 43. The method as claimed in claim 29, wherein thefreezing step comprises freezing at about −45° C. for about 60 minutes.44. The method as claimed in claim 29, wherein the primary drying stepcomprises ramping at about +0.5° C./minute to 1.0° C./minute to achieveshelf temperature of about −32° C., holding for about 600 minutes to1800 minutes.
 45. The method as claimed in claim 29, wherein thesecondary drying step comprises ramping at about +0.5° C./minute to 1.0°C./minute to achieve shelf temperature of about +25° C., holding forabout 360 minutes to 600 minutes. 46.-51. (canceled)
 52. A kitcomprising: a. a first container containing an immunogenic composition,said composition comprising: i. one or more live attenuated dengue (DEN)virus; ii. sucrose 3 to 6% (w/v); and iii. glycine 3 to 6% (w/v); and b.a second container containing an aqueous solution selected from salineor WFI (water for injection) for the reconstitution of the lyophilized(freeze-dried) immunogenic composition.